Best way to determine dead volume of labware for Hamilton aspiration

Hi all,

I am trying to determine the dead volume of various labware and was wondering what the best method was? Initially I thought I could go with using Hamilton MAD (monitored air displacement) but after some testing it does not seem accurate enough for reporting true dead volume i.e… not accurate for under 50ul

What is the most empirical way for determining dead volume of a container for a Hamilton instrument aspiration?

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@cwehrhan - I wouldn’t say there is a one-size-fits-all solution here. Dead volume requirements are situational, and vary based on several factors:

  • Liquid handling parameters (liquid following and tip submerge depth are most significant)
  • Reagent surface tension
  • Labware geometry and coatings
  • Properly defined definitions for used labware

Even the same combination of labware and reagent can have different dead volume needs when changing the liquid handling used. Do you define dead volume by the amount of liquid required to be in the tube to not trigger an insufficient liquid error? Or by which an amount that allows for complete aspirations from a height just above the bottom of a well? An aspiration with varying liquid detection, following, tip submerge depths, and error handling approaches will yield different dead volume needs depending on requirements.

For different labware and reagent combinations, the reagent may tend to bead up or coalesce into discrete regions, resulting in a non uniform liquid level as the volume approaches lower limits at varying rates. Geometry of the bottom of the labware as well as surface tension will have the greatest impact here.

Additionally, it is important to ensure your labware definition matches your tube/well geometry, and the deck z coordinate is accurate so the system can calculate following rates and remaining volume in a way that reflect reality (if using liquid following). When observing the tip move down in z as it aspirates, it it keeping a consistent distance between the bottom of the tip and the liquid surface? If not, the container definition needs to be adjusted.

All that said, the best thing to do is test using representative reagents (if possible) and liquid handling parameters which will be used in the method, for critical reagents. You can aspirate set amounts of a known volume, and re-dispense back into the container to characterize a robust dead volume limit.



I second that it depends on how you define dead volume. Is it the reachable volume in a tube? Or the min. volume to guarantee for robust workflow?

As @NickHealy_Hamilton mentioned, there are many aspects to consider. Don’t forget about imprecision of the x/y position of the source target: can you really ensure to aspirate from the very center? It depends on the container shape, e.g. has a higher impact with plates. Also keep workflow dependent factors like evaporation in mind. Imprecision of LLD combined with cavity diameter also adds todead volume. All of the mentioned aspects require additional volume.

In most cases it may be hard to calculate the dead volume precisely, so I would recommend to evaluate it empirically: estimate the expected dead volume following the above considerations, load the containers of interest, dispense various volumes around the estimated volume into (like e.g. 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3 times) and try to aspirate using your original workflow. The required dead volume is reached if none of the replicates throws an aspiration error. I recommend to apply 10 replicates per level and not to reuse positions (like aspirate from and dispense into the equal position multiple times) due to losses of liquid, e.g. on the tip surface if you eject the tips, the filling volume will decrease over time.

Hope that helps


The way I do it is to start with the manufacturer specs of the residual volume. It’s usually a very good benchmark to start off with. So for example if a flat bottom corning plate is 50uL residual volume. I would prepare 55uL and then aspirate from the bottom of the plate (or whichever liquid class you use) and do 1uL increments - loop this 55 times - onto an accuracy assay - such as an Artel.

The moment it fails is the point you can accurately determine your residual volume.

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